The aim of this proposal is to test and apply a new technique (fluorescence lifetime heterogeneity analysis) for identifying the existence of microheterogeneity in membrane structure. The power of lifetime heterogeneity analysis is its potential sensitivity for detecting short-lived membrane microdomains which do not appear as thermodynamically distinct phases. The applicability of this technique will first be evaluated in simple model membrane systems in which structural heterogeneity is well established. Then, as initial applications, this method will be used to test two hypotheses stemming from work of the previous grant period: 1) that a trans-membrane protein both orders and disorders its local lipid environment, and 2) that cholesterol induces calorimetrically undetectable local domains in fluid lipid bilayers. New applications will use lifetime analysis to detect microstructures thought to be associated with two key cellular processes, membrane fusion and phospholipid trans-membrane migration. In addition to fluorescence lifetime analysis, appropriate thermodynamic techniques (scanning calorimetry, steady-state probe fluorescence, chemical labeling, freeze-fracture electron microscopy) will be used to examine bilayer structural features associated with these events in well-defined, model systems. In the case of polyethyleneglycol-induced membrane fusion, these measurements will test the hypothesis that different structural characteristics are associated with two crucial and previously inseparable intermediate states in the fusion process: bilayer close apposition and bilayer destabilization. In the case of cation-induced phosphatidylglycerol transbilayer redistribution, results will test two hypotheses: 1) transbilayer redistribution involves a two-component interaction between a metal ion and a lipid bilayer and 2) this interaction induces inverted micelle-like local structures in the lipid bilayer.